The rate of protein-protein association plays critical roles in many fundamental biological processes ranging from enzyme catalysis/inhibition to regulation of immune response by cytokines. Our long-term objectives are to reliably predict association rates given just the structures of protein complexes and elucidate the mechanisms of protein association. We have fully developed a transient-complex theory and demonstrated its predictive power in a comprehensive study of protein-protein association. The transient-complex theory presents unique opportunities to uncover molecular bases for wide variations in association rates among proteins and design proteins with desired binding properties, which we exploit in this project. Specific Aim 1 is to develop a new implementation of the transient-complex theory. The new developments will account for contributions of hydrophobic interactions and configurational entropy and allow for modeling of protein conformational sampling in the transient complex. In Specific Aim 2, the transient-complex theory will be used to systematically design mutations that will turn slow-binding complexes into fast-binding ones and to quantitatively rationalize large differences in association rate between related proteins. Test systems will include complexes of the Wiskott-Aldrich Syndrome protein with two homologous Rho GTPases. Some of the designed mutants will be tested experimentally through collaboration. In Specific Aim 3 we will apply the transient-complex theory to protein-RNA complexes. Targeting distinct sites for binding ribonuclease inhibitor and RNA, variants of ribonuclease A that bind the former ineffectively but the latter efficiently will be designed as potential anti-cancer therapeutics. Together, these applications will demonstrate the ability to control protein functions by manipulating protein interactions and yield valuable insight on the biological roles of protein association rates. PUBLIC HEALTH RELEVANCE: The proposed research will have both indirect and tangible benefits to public health. It will advance fundamental understanding of a broad range of biological processes, such as the stimulation of actin polymerization by the Wiskott-Aldrich Syndrome protein and the discrimination of Trna between cognate and non-cognate aminoacyl-tRNA synthetases (a step critical for the fidelity of the translation of the genetic code). The research may also benefit directly the design of asthma and cancer therapeutics.